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Marcia J Kieliszewski - One of the best experts on this subject based on the ideXlab platform.
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Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant
Plants, 2015Co-Authors: Yuning Chen, Dening Ye, Tui Ray, Alexandra Frye, M C Cannon, Andrew J. Mort, Michael A. Held, Prasenjit Saha, Marcia J KieliszewskiAbstract:Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding Extensin 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes.
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Changes in cell wall properties coincide with overexpression of Extensin fusion proteins in suspension cultured tobacco cells
PLoS ONE, 2014Co-Authors: Li Tan, Allison Arter, Yunqiao Pu, Liwei Chen, Sivakumar Pattathil, Michael G Hahn, Jin Qian, Utku Avci, Arthur J Ragauskas, Marcia J KieliszewskiAbstract:Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-Extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the Extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified Extensins may be a route to engineer plants for bioenergy and biomaterial production.
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Role of the Extensin superfamily in primary cell wall architecture.
Plant physiology, 2011Co-Authors: Derek T A Lamport, Yuning Chen, Marcia J Kieliszewski, M C CannonAbstract:Nearly two centuries of progress have established the major components of the plant cell wall, a composite that includes interpenetrating networks of cellulose (Payen, 1838; Schulze, 1891), microfibrils (Frey-Wyssling et al., 1948; Preston et al., 1948), pectin (Braconnot, 1825) and lignin (Payen, 1838). However, only over the last five decades has a relatively minor hydroxyproline-rich structural glycoprotein component emerged with essential roles in building and maintaining the growing primary cell wall. Here we highlight unique advances of each decade from the initial discovery of hydroxyproline (Hyp) in cell walls to the current definition of Extensins as self-assembling amphiphiles that generate scaffolding networks, and where acid-base interaction - Extensin pectate - may template assembly of the pectic matrix. Subsequent polymerization toughens up the wall as networks resisting both microbial and mechanical stress. At each stage we explore hypotheses arising from synthesis of emerging data with focus on structure. This review celebrates the 50th birthday of Extensin.
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Di-isodityrosine is the intermolecular cross-link of isodityrosine-rich Extensin analogs cross-linked in vitro
Journal of Biological Chemistry, 2004Co-Authors: Michael A. Held, Abdolreza Kamyabi, Elena Shpak, Michael O'hare, Li Tan, Marcia J KieliszewskiAbstract:Extensins are cell wall hydroxyproline-rich glycoproteins that form covalent networks putatively involving tyrosyl and lysyl residues in cross-links catalyzed by one or more Extensin peroxidases. The precise cross-links remain to be chemically identified both as network components in muro and as enzymic products generated in vitro with native Extensin monomers as substrates. However, some Extensin monomers contain variations within their putative cross-linking motifs that complicate cross-link identification. Other simpler Extensins are recalcitrant to isolation including the ubiquitous P3-type Extensin whose major repetitive motif, Hyp)(4)-Ser-Hyp-Ser-(Hyp)(4)-Tyr-Tyr-Tyr-Lys, is of particular interest, not least because its Tyr-Tyr-Tyr intramolecular isodityrosine cross-link motifs are also putative candidates for further intermolecular cross-linking to form di-isodityrosine. Therefore, we designed a set of Extensin analogs encoding tandem repeats of the P3 motif, including Tyr --> Phe and Lys --> Leu variations. Expression of these P3 analogs in Nicotiana tabacum cells yielded glycoproteins with virtually all Pro residues hydroxylated and subsequently arabinosylated and with likely galactosylated Ser residues. This was consistent with earlier analyses of P3 glycopeptides isolated from cell wall digests and the predictions of the Hyp contiguity hypothesis. The tyrosine-rich P3 analogs also contained isodityrosine, formed in vivo. Significantly, these isodityrosine-containing analogs were further cross-linked in vitro by an Extensin peroxidase to form the tetra-tyrosine intermolecular cross-link amino acid di-isodityrosine. This is the first identification of an inter-molecular cross-link amino acid in an Extensin module and corroborates earlier suggestions that di-isodityrosine represents one mechanism for cross-linking Extensins in muro.
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Matrix assisted laser desorption/ionization time-of-flight mass spectrometry of tomato Extensin monomers and potato lectin
Phytochemistry, 1997Co-Authors: Marcia J Kieliszewski, Ron OrlandoAbstract:Abstract Extensins are basic hydroxyproline-rich glycoproteins (HRGPs) of the primary cell wall. They exist mainly as an insoluble network, but also as soluble precursors which are readily eluted from intact cultured cells by dilute salt solutions. Although generally regarded as monomeric, these soluble network precursors exhibit anomalous behaviour on gel electrophoresis and gel filtration, which has recently led to an alternative view of soluble Extensin as globular-like aggregates of covalently crosslinked monomers. This discrepancy needs to be resolved as it impinges directly on mechanisms of wall assembly. Therefore, using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) we measured the molecular masses of P1 and P2, two highly glycosylated soluble Extensins from tomato. P1 was 89.7 kDa while P2 was 132.1 kDa. MALDI-TOF MS control experiments with the related HRGP, potato lectin, showed that it occurred as a mixture of monomers and dimers. This excludes the likelihood of oligomer disruption and confirms earlier conclusions that soluble Extensins exist mostly as monomers.
Derek T A Lamport - One of the best experts on this subject based on the ideXlab platform.
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Detection of Glycosylated and Deglycosylated Extensin
2016Co-Authors: Theresa A. Conrad, Derek T A Lamport, Raymond HammerschmidtAbstract:A competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the rapid quantitation of the glycosylated and deglycosylated forms of the monomeric soluble Extensin precursor subunits Pl and P2. A log-linear response range for each kind of precursor in the competition curve was between 0.01 and 100 nanograms per milliliter. Extensin is a plant cell wall-bound hydroxyproline-rich glycoprotein. Two different soluble forms of Extensin were recently isolated from salt eluates of intact tomato cell suspension cultures (12). These HRGPs3 were shown to be different in amino acid composition. These two soluble Extensins, P1 and P2, are monomeric precursors to insoluble network Extensin. Although the evidence is correlative, Extensin may function as part of the resistance mechanism against certain biological stresses. Extensin accumulates in cell walls in response to stress stimuli such as wounding (14), pathogen attack (2, 3, 8, 9), and heat shock (13), but the precise role of this response is not known. Key to understanding the role of Extensin in these responses is the ability
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Role of the Extensin superfamily in primary cell wall architecture.
Plant physiology, 2011Co-Authors: Derek T A Lamport, Yuning Chen, Marcia J Kieliszewski, M C CannonAbstract:Nearly two centuries of progress have established the major components of the plant cell wall, a composite that includes interpenetrating networks of cellulose (Payen, 1838; Schulze, 1891), microfibrils (Frey-Wyssling et al., 1948; Preston et al., 1948), pectin (Braconnot, 1825) and lignin (Payen, 1838). However, only over the last five decades has a relatively minor hydroxyproline-rich structural glycoprotein component emerged with essential roles in building and maintaining the growing primary cell wall. Here we highlight unique advances of each decade from the initial discovery of hydroxyproline (Hyp) in cell walls to the current definition of Extensins as self-assembling amphiphiles that generate scaffolding networks, and where acid-base interaction - Extensin pectate - may template assembly of the pectic matrix. Subsequent polymerization toughens up the wall as networks resisting both microbial and mechanical stress. At each stage we explore hypotheses arising from synthesis of emerging data with focus on structure. This review celebrates the 50th birthday of Extensin.
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Isolation of pl 4.6 Extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular cross-link site.
The Plant journal : for cell and molecular biology, 1996Co-Authors: L S Schnabelrauch, H Alizedeh, Brad L. Upham, Marcia J Kieliszewski, Derek T A LamportAbstract:Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network Extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose-6) gel filtration assay of cross-linked Extensin oligomers, a pl 4.6 Extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic Extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 Extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 Extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5. Assayed with 12 different Extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-Extensins and non-cross-linking or NCL-Extensins. CL-Extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated. However, the simplest motif common to CL-Extensins but absent from NCL-Extensins was Val-Tyr-Lys. Thus, peroxidative coupling of Extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine.
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Isolation of pl 4.6 Extensin peroxidase from tomato cell suspension cultures and identification of Val—Tyr—Lys as putative intermolecular cross‐link site
The Plant journal : for cell and molecular biology, 1996Co-Authors: L S Schnabelrauch, H Alizedeh, Brad L. Upham, Marcia J Kieliszewski, Derek T A LamportAbstract:Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network Extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose-6) gel filtration assay of cross-linked Extensin oligomers, a pl 4.6 Extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic Extensin peroxidase (pl 4.6) on DEAE-Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 Extensin as substrate and 60 microM H2O2 as co-substrate, at 23 degrees pl 4.6 Extensin peroxidase gave a Km of 0.22 mM P1 and a Vmax 0f 70 mumol P1 cross-linked min-1mg-1 enzyme, at the optimum pH 5.5. Assayed with 12 different Extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-Extensins and non-cross-linking or NCL-Extensins. CL-Extensins contained the X-Hyp-Val-Tyr-Lys motif and were also highly glycosylated. However, the simplest motif common to CL-Extensins but absent from NCL-Extensins was Val-Tyr-Lys. Thus, peroxidative coupling of Extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine.
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Extensin repetitive motifs functional sites post translational codes and phylogeny
Plant Journal, 1994Co-Authors: Marcia J Kieliszewski, Derek T A LamportAbstract:Homologous hydroxyproline-rich glycoproteins (HRGPs) of the plant extracellular matrix include Extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein (GAGP), arabinogalactan-proteins (AGPs), and chimeric proteins such as potato lectin which contain an Extensin module fused to a lectin. The key to the role of HRGPs in cell wall self-assembly and cell extension lies in their chemistry, which is dependent on extensive post-translational modifications (PTMs): hydroxylation, glycosylation, and cross-linking. Repetitive peptide motifs characterize HRGPs. One or more repetitive peptide motifs and their variants, singly or in combination, may constitute functional sites involved in various aspects of cell wall assembly, as follows: (i) X-Hypn including Ser-Hyp4 (arabinosylation site, molecular rigidity, and reptation). (ii) Pro-Hyp-Val-Tyr-Lys and variants (putative intermolecular cross-links, adhesion, cohesion, and possible beta-turns). (iii) Tyr-X-Tyr-Lys (intramolecular isodityrosine [IDT] cross-links increase molecular rigidity and hydrophobicity). (iv) (Glyco)peptide palindromes (centrosymmetric domains: putative self-assembly nucleation sites). (v) Ionic interaction sites (protein-protein and protein-carbohydrate cross-links). (vi) Hyp and Ser glycosylation sites (enhance conformational stability and molecular recognition). (vii) Extensin modules in chimeric proteins (e.g. solanaceous lectins). Rules for the post-translational modifications are emerging: (i) Hydroxylation of proline residues may depend on multiple, sequence-specific prolyl hydroxylases rather than on a single (polyproline-II) conformation-dependent enzyme. Furthermore, Lys-Pro, Tyr-Pro, and Phe-Pro are not hydroxylated, while Pro-Val is always. (ii) Contiguity of Hyp residues probably determines the extent of Hyp glycosylation, blocks of tetrahydroxyproline (Hyp4) being the most highly arabinosylated, while single non-contiguous Hyp residues are rarely arabinosylated, although they are likely attachment sites for the larger arabinogalactan substituents of gum arabic glycoprotein and arabinogalactan-proteins. (iii) While intramolecular cross-links involve IDT, unidentified intermolecular cross-links most likely involve the Val-Tyr-Lys motif (perhaps also Val-Lys-Pro-Tyr-His-Pro), probably as an adduct between Tyr and Lys catalyzed in vitro by a pI 4.6 Extensin cross-linking peroxidase. Thus, we can classify HRGPs functionally as either cross-linking or non-cross-linking, i.e. CL- or NCL-Extensins. Their protistan origin obscures the phylogenetic affinities of a single Extensin-HRGP family due to their sequence divergence. We propose a phylogenetic series ranging from the minimally glycosylated basic RPRPs to the highly glycosylated acidic AGPs. Furthermore, based on similarities between dicots and gymnosperm Extensins, and their marked difference from graminaceous monocot Extensins, graminaceous monocot and dicot lines may have diverged as early as the progymnosperms.(ABSTRACT TRUNCATED AT 400 WORDS)
Allan M. Showalter - One of the best experts on this subject based on the ideXlab platform.
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Extensin and Arabinogalactan-Protein Biosynthesis: Glycosyltransferases, Research Challenges, and Biosensors
Frontiers in plant science, 2016Co-Authors: Allan M. Showalter, Debarati BasuAbstract:Recent research, mostly in Arabidopsis thaliana, has led to the identification and characterization of the glycosyltransferases responsible for the biosynthesis of two of the most functionally important and abundant families of plant cell wall proteins, Extensins and arabinogalactan-proteins. Extensin glycosylation involves monogalactosylation of serine residues by O--serine galactosyltransferase and the addition of oligoarabinosides one to five arabinose units in length to contiguous hydroxyproline residues by a set of specific arabinosyltransferase enzymes, which includes hydroxyproline O-β-arabinosyltransferases, β-1,2-arabinosyltransferases, and at least one α-1,3-arabinosyltransferase. AGP glycosylation, however, is much more complex and involves the addition of large arabinogalactan polysaccharide chains to non-contiguous hydroxyproline residues. These arabinogalactan chains are composed of -1,3-galactan backbones decorated with -1,6-galactose side chains that are further modified with -arabinose as well as other sugars, including -(methyl)glucuronic acid, -rhamnose, and -fucose. Specific sets of hydroxyproline O--galactosyltransferases, -1,3-galactosyltransferases, -1,6- galactosyltransferases, -arabinosyltransferases, -glucuronosyltransferases, -rhamnosyltransferases, and -fucosyltransferases are responsible for the synthesis of these complex structures. This mini-review summarizes the EXT and AGP glycosyltransferases identified and characterized to date along with corresponding genetic mutant data, which addresses the functional importance of EXT and AGP glycosylation. In one case, genetic mutant data indicate that the carbohydrate moiety of arabinogalactan-proteins may serve as an extracellular biosensor or signal for normal cellular growth. Finally, future research challenges with respect to understanding the function of these enzymes more completely and discovering and characterizing additional glycosyltransferases responsible for Extensin and arabinogalactan-protein biosynthesis are also discussed.
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Potato lectin: a modular protein sharing sequence similarities with the Extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors).
The Plant journal : for cell and molecular biology, 1994Co-Authors: Marcia J Kieliszewski, Allan M. Showalter, J F LeykamAbstract:Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp- Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the 'P3' type Extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp- suggesting common evolutionary origins for the Extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and Extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.
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Isolation and characterization of two wound-regulated tomato Extensin genes
Plant Molecular Biology, 1992Co-Authors: Jin Zhou, Dominique Rumeau, Allan M. ShowalterAbstract:Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library by in situ plaque hybridization with Extensin DNA probes. Tom J-10 encoded an Extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded Extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro_4-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro_4-Tyr_3-Lys-Ser-Pro_4-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 Extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an Extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded Extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro_4 repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro_4-Ser-Pro-Ser-Pro_4-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 Extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.
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Molecular details of tomato Extensin and glycine-rich protein gene expression
Plant Molecular Biology, 1992Co-Authors: Allan M. Showalter, Adrian D. Butt, Sungham KimAbstract:In a recent publication (Plant Molecular Biology 16: 547–565 (1991)) Showalter et al. described the isolation and initial characterization of fifteen Extensin and Extensin-like tomato cDNAs. These cDNAs were determined to fall into five distinct classes; class I and II clones encoded Extensins, class III and V clones encoded glycine-rich proteins (GRPs), and class IV clones encoded a portion of a GRP sequence on one DNA strand and a portion of an Extensin sequence on the other DNA strand. In this publication, a more detailed analysis of the expression of these cDNA classes was performed with respect to wounding in various tomato organs, development, kinetics and systemic extent of the wound response, ethylene treatment, abscisic acid (ABA) treatment, and drought stress by using RNA gel blot hybridizations. In general, Extensin gene expression was readily detected in stems and roots, but not in leaves. With both class I and II Extensin cDNA probes, wound-induced accumulation of mRNA in stems was first detected between 4 and 8 h after wounding with maximal accumulation occurring after 12 h. Moreover, these Extensin wound responses were detected locally at the wound site but not systemically. Expression of the class III GRP was largely limited to wounded stem tissue. Initial detection and maximal accumulation of the class III GRP mRNA was similar to the Extensins mRNAs; however, this GRP wound response occurred both locally and systemically. Additionally, abscisic acid treatment and drought stress resulted in the marked accumulation of the class III GRP mRNA in tomato stems, but did not alter the expression of the other cDNA classes. In contrast, expression of the class V GRP occurred in stems and roots and to a lesser extent in leaves and decreased in response to wounding over a 24 h time period. The class V GRP wound response was further characterized by an early, transient accumulation of mRNA occurring 2–4 h after wounding in stems and by its local nature.
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Tomato Extensin and Extensin-like cDNAs: structure and expression in response to wounding.
Plant molecular biology, 1991Co-Authors: Allan M. Showalter, Dominique Rumeau, Jin Zhou, Susan G. Worst, Joseph E. VarnerAbstract:Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in λgt10 and screened by in situ plaque hybridization with a tomato Extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an Extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an Extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2–6-Tyr-Pro and(Gly)2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an Extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2–5-Arg repeats.
Prakash M. Dey - One of the best experts on this subject based on the ideXlab platform.
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Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity.
Planta, 1997Co-Authors: Prakash M. Dey, M. D. Brownleader, A. T. Pantelides, M. Trevan, J. J. Smith, G. SaddlerAbstract:Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato Extensin peroxidase and H2O2. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immuno-sorbent assay of the B isoform, using tomato Extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato Extensin monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457–469). The amino acid and carbohydrate compositions were similar to those of tomato Extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to Extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of Extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possesses an extended polyproline II helix conformation with no evidence of α- helix or β- sheet secondary structure. In conclusion, we refer to this HRGP as potato Extensin. As proposed for other Extensins, potato Extensin is likely to play a role in cell wall architecture and plant disease resistance.
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Purification and Partial Characterization of Tomato Extensin Peroxidase.
Plant physiology, 1995Co-Authors: M. D. Brownleader, M. Trevan, Naziha Ahmed, Martin F. Chaplin, Prakash M. DeyAbstract:Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as Extensin, in the cell wall. The insolubilization process (cross-linking between soluble Extensin precursor molecules) is catalyzed by Extensin peroxidases. We have ionically eluted Extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble Extensin. The molecular weight (34,000–37,000), amino acid composition, and isoelectric point (9.0) of the Extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble Extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles Extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except Extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and Extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato Extensin was used to demonstrate Extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links Extensin has been purified and may have a role in plant defense.
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Purification of Extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture.
Planta, 1993Co-Authors: Michael D. Brownleader, Prakash M. DeyAbstract:Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of β-l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both Extensin precursor and Extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native Extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted Extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, Extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the Extensin preparation. Evidence that the purified protein was Extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)4 — within the N-terminal end of the protein. Treatment of Extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified Extensin was similar to those reported in the literature. The cross-linking of Extensin in vitro upon incubation with Extensin peroxidase and exogenous H2O2 was characteristic of other reported Extensins. Furthermore, Mono-S ion-exchange FPLC of native Extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of Extensin A and Extensin B were found to be similar to each other and both Extensins were cross-linked in vitro by Extensin peroxidase.
Azeddine Driouich - One of the best experts on this subject based on the ideXlab platform.
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Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization
Annals of Botany, 2020Co-Authors: Romain Castilleux, Barbara Plancot, Bruno Gugi, Agnes Attard, Corinne Loutelierbourhis, Benjamin Lefranc, Eric Nguemaona, Mustapha Arkoun, Jeanclaude Yvin, Azeddine DriouichAbstract:BACKGROUND AND AIMS: Extensins are hydroxyproline-rich glycoproteins thought to strengthen the plant cell wall, one of the first barriers against pathogens, through intra- and intermolecular cross-links. The glycan moiety of Extensins is believed to confer the correct structural conformation to the glycoprotein, leading to self-assembly within the cell wall that helps limit microbial adherence and invasion. However, this role is not clearly established. METHODS: We used Arabidopsis thaliana mutants impaired in Extensin arabinosylation to investigate the role of Extensin arabinosylation in root-microbe interactions. Mutant and wild-type roots were stimulated to elicit an immune response with flagellin 22 and immunolabelled with a set of anti-Extensin antibodies. Roots were also inoculated with a soilborne oomycete, Phytophthora parasitica, to assess the effect of Extensin arabinosylation on root colonization. KEY RESULTS: A differential distribution of Extensin epitopes was observed in wild-type plants in response to elicitation. Elicitation also triggers altered epitope expression in mutant roots compared with wild-type and non-elicited roots. Inoculation with the pathogen P. parasitica resulted in enhanced root colonization for two mutants, specifically xeg113 and rra2. CONCLUSIONS: We provide evidence for a link between Extensin arabinosylation and root defence, and propose a model to explain the importance of glycosylation in limiting invasion of root cells by pathogenic oomycetes.
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Cell wall Extensins in Root-microbe Interactions and Root Secretions
Journal of Experimental Botany, 2018Co-Authors: Romain Castilleux, Barbara Plancot, Marc Ropitaux, Alexis Carreras, Jérôme Leprince, Isabelle Boulogne, Marie-laure Follet-gueye, Zoë Popper, Azeddine Driouich, Maïté VicréAbstract:Extensins are cell wall glycoproteins, belonging to the Hydroxyproline-Rich GlycoProtein (HRGP) family, which are involved in many biological functions, including plant growth and defence. Several reviews have described the involvement of HRGPs in plant immunity but little focus has been given specifically to cell wall Extensins. Yet, a large set of recently published data indicates that Extensins play an important role in plant protection, especially in root-microbe interactions. Here, we summarize the current knowledge on this topic and discuss the importance of Extensins in root defence. We first provide an overview of the distribution of Extensin epitopes recognised by different monoclonal antibodies among plants and discuss the relevance of some of these epitopes as markers of the root defence response. We also highlight the implication of Extensins in different types of plant interactions elicited by either pathogenic or beneficial microorganisms. We then present and discuss the specific importance of Extensins in root secretions as these glycoproteins are not only found in the cell walls but are also released into the root mucilage. Finally, we propose a model to illustrate the impact of cell wall Extensin on root secretions.
